Since the pioneering work of Merrifield (Merrifield, R. B. (1963), J. Am. Chem. Soc., 85, 2149–2153) on polystyrene (2% divinylbenzene cross-linked) as solid support for peptide synthesis, several improvements on the nature of the solid support were brought about to meet special needs of new organic chemistry. Through the years, most of the work done in that field has been focused on peptide synthesis.
Several polyamide resins (Kanda et al., (1991), Int. J. Peptide Protein Res., 38, 385–391) for solid phase peptide synthesis have been developed since the '80. The amide bonds of the polymer are the same as those found in peptides. Consequently, peptide chemistry can be performed in a polarity environment which is similar to that of peptides and that improves chemical yields and peptide purity. PEPSYN (Arshady et al., (1981), J. Chem. Soc. Perkin Trans., 529–537), PEPSYN K (Atherton et al., (1981), J. Chem. Soc. Chem. Commun., 1151–1152), and Polyhipe (Small et al., (1989), J. Chem Soc. Chem Commun., 1589–1591), can be mentioned as other types of solid supports for peptide chemistry which were developed during the period 1981–1989.
The first (and probably the most famous) polystyrene-PEG (polyethylene glycol) hybrid resin is the one developed by Bayer known as the TentaGel® and disclosed in U.S. Pat. No. 4,908,405 and in Bayer E. (1991), Angew. Chem. Int. Ed. Engl. 30, 113–129. The matrix is made by coupling tetraethylene glycol (TTEG) with chloromethylated polystyrene. A high molecular weight PEG is then introduced into the polymer by reacting ethylene oxide with the potassium salt of PS-TTEG polystyrene-TTEG). That method has been proposed to give higher yields as a result of the further reaction with ethylene oxide. This polymer offers a good compromise between the mechanical properties of polystyrene and desired amphiphile behaviour with good swelling. The Bayer Patent also includes the use of cross-linked acrylates and methacrylates which are functionalized with hydroxy groups, hydroxypolystyrene and polyvinyl alcohol as starting materials. In this manner, it is possible to obtain (with ester bonds attached to the polymer), benzylic and secondary ethers. One of the drawbacks of that method is the possible cross-linking of TTEG (under basic conditions) between two chloromethylated benzene rings of the polymer network.
Meldal in Tetrahedron Lett., 33, 3077–3080 (1992) and in U.S. Pat. No. 5,352,756, as well as Renil et al., in Tetrahedron Lett, 36, 4647–4650, (1995) proposed a new polar matrix called PEGA. That polymer contains PEG or PPG (long chains) cross-linker bearing acrylamide moieties which are copolymerized with other methacrylic derivatives. Some of them are used as linkers for solid phase synthesis purposes. The choice of long chain PEG or PPG permits the passage of peptidic molecules through its network. The amide bonds found in that polymer are appropriate for solid phase peptidic synthesis.
Lee, in U.S. Pat. No. 5,466,758 and Park et al., (1997), Tetrahedron Lett., 38, 591–594 demonstrated the versatility of a process for the production of polystyrenes having a β-hydroxy group and polyglycol-grafted thereon. Based on the work of and Milstein (Milstein, N. (1968), J. Heterocycl. Chem., 5, 337–338) and Suga (Nakajima et al., (1969), Tetrahedron Lett., 38, 591–594 and citations therein), it is known to submit propylene oxide to Friedel-Crafts reactions with benzene and other aromatic compounds to give such β-hydroxy groups. Once the hydroxylated polystyrene is sythesized, the later reacts with ethylene oxide under basic conditions to give a PEG-polystyrene with various loading of terminal hydroxy groups. Lee established the stability of his new PEG-polystyrene matrix by several acidic treatments encountered in peptidic chemistry without any degradation, while conventional TentaGel® is degraded.
ArgGel® (Labadie et al., WO 97/27226, 1996) was developed during the same time. This Merrifield based resin has a better stability than its predecessor. Indeed, no benzyl ethers are present in the matrix through the use of a malonate derivative linked to the benzylic position. Subsequently, the diester is reduced to a diol and polymerized with ethylene oxide to give a stable PEG-polystyrene matrix.
Barany (Kempe et al., (1996) J. Am. Chem. Soc. 118, 7083–7093, and U.S. Pat. No. 5,910,554 refers to a highly cross-linked polymeric support called CLEAR® which is based on the copolymerization of tertiary cross-linkers containing polyethylene glycol (PEG) or polypropylene glycol (PPG) with several vinyl and allyl derivatives. The main application of that solid support is in the field of peptide synthesis. In the normal peptide chemistry conditions encountered, the integrity of the matrix is preserved. On the other hand, Tuncel, in Colloid Polym. Sci., 278, 1126–1138 (2000) described the synthesis of such swellable matrix based on PEG methacrylates, said matrix having a controlled hydrophilicity and functionnality.
Meldal has released two different approaches, as discussed below, to reach a backbone made from primary and/or secondary ether bonds with alcohol residues on which organic chemistry can be performed.
POEPOP (polyoxyethylene-polyoxypropylene) Renil et al., (1996), Tetrahydron Lett., 37, 6185 –6188) is made from epichlorohydrin and polyethylene glycol (PEG) to produce an epoxy material. The latter product is then polymerized with potassium t-butoxide. By a simple adjustment of the amount of epoxy residues on the original PEG 1500, a polymer is formed with a definite amount of alcohol to be derivatized. In that feature, a mixture of secondary and primary ether bond are formed with strong chemical resistance and good physical properties.
SPOCC (solid phase organic and combinatorial chemistry) (Rademann et al., (1999), J. Am. Chem. Soc., 121, 5459–5466 and Meldal et al., (2000), WO 00/18823) bas been proposed to obtain a backbone with ether bonds exclusively. In the same way, the polymer is formed of a high molecular weight PEG, but with oxetane end groups. The final product is obtained by a cationic ring-opening polymerization with BF3Et2O in a silicon oil media. The major advantage of that matrix is its chemical stability from its primary ether bonds that has never been reached up to this day.
POEPOP and SPOCC are manufactured under expensive conditions (silicon oil) to obtain standard beaded polymer making its commercial production non attractive as conventional on polymerization methods with vinyl derivatives.
Bayer (Mutter et al., (1971), Angew. Chem., 83, 883; Angew. Chem. Int. Ed. Engl. 12, 811 (1971)) introduced the concept of liquid-phase chemistry where a high molecular weight PEG (one end optionally capped) is functionnalized to directly perform peptide chemistry thereon. This low cost product has the advantage of being compatible with practically all solvents used in organic chemistry except diethyl ether. The latter is used as the precipitation media which permit the filtration of the PEG resin at the end of the synthesis. The fact that PEG is solubilized in the reaction media ensures the accessibility of the reagents dissolved in the solvent to the reactive functionalities which are present on the polymer. Unfortunately, when ethers (Et2O or MTBE methyl tert-butyl ether) or alcohols (EtOH or cold i-PrOH) are used to precipitate the polymer, some impurities in the reaction mixture can also be precipitated. Even considering the low cost of PEG resins, that feature considerably reduces the attractiveness of the linear PEG family in solid phase peptide chemistry. This approach had been well documented during the 70′ and 80′. Janda (Wenworth et al., (1999, Chem. Comm., 1917–1924) and many others (Annunziata et al., (2001), J. Org. Chem., 66, 3160–3166 and citations therein) pursue this idea since.
In a different approach, Janda used several styrenic-etheral crosslinked agents which are copolymerized with styrene (Janda et al., (1999), Tetrahedron Lett., 40, 6329). These crosslinked agents obtained from polytetrahydrofuran and 4-chloromethylstyrene or 4-hydroxystyrene, provide alternatives for divinylbenzene. Janda obtained good polymers with impressive swelling properties. This work showed how polystyrene can be upgraded with a little tuning of the crosslinked agent. Even though ether bonds are introduced in the polymer network, the hydrophobicity of styrene is still present.
Meldal (Renil et al., (1996), Tetrahedron Lett., 37, 6185–6188) used high molecular weight (Mw 1500) PEG for the same experience with 4-chloromethylstyrene and 3-chloropropylstyrene (Buchardt et al., (1998); Tetrahedron Lett., 39, 8695–8698; Meldal et al., (2000), WO 00/18823). Wilson (Wilson et al., (1998), J. Org. Chem., 63, 5094–5099) employed shorter PEGs with 4-chloromethylstyrene (from ethylene to hexaethylene glycol) to obtain different polymers.
Roice (Roice et al. (1999), Macromol., 32, 8807–8815) proposed a butanediol dimethacrylate—styrene flexible copolymer for solid phase peptide synthesis. Under normal reaction conditions, no degradation had been observed. Unfortunately, esters residues are susceptible to degradation in strong acidic and basic conditions that can be encountered in organic chemistry. Other PEG-PS copolymers containing ester residues have been made in a similar way by using different monomers such as tetraethylene glycol and hexanediol diacrylates (Hellerman et al., (1983), Makrom. Chem., 184, 2603; Renil et al., (1994), Tetrahedron, 50, 6681; Zalipsky et al., (1994), React. Polym., 22, 243; Varkey et al., (1998), J. Peptide Res., 51, 49).
Meldal (Groth et al., (2000), WO 00/18823) proposed a new PEG based polymer named HYDRA. The polymer is cross-linked with tris (2-aminoethyl)amine on the PEG-aldehyde derivative via a reductive amination optionally containing hydroxyl functionalities. The residual amines or alcohols are therefore useful for anchoring several types of linkers. Unfortunately, the making of the polymer in a beaded form is impossible.
Other useful references on many other solid supports are available elsewhere (Meldal et al., (1997), Methods in Enzymology, 289, 83–104, Academic Press, N.Y.).
It is an object of the present invention to provide a new family of polymeric solid supports based on a polyethylene glycol or polypropylene glycol matrix.
It is another object of the present invention to provide a method for the synthesis of such polymeric solid supports.
It is another object of the present invention to provide polymeric solid supports that can be used for the solid phase synthesis of peptides, oligonucleotides, oligosaccharides and in combinational and traditional organic chemistry.
It is another object of the present invention to provide resins that can be used in liquid phase synthesis, chromatography, for scavenging purposes, and for protein and reagents immobilization.
It is another object of the present invention to provide a polymeric matrix based on the copolymerization of a polyethylene glycol or polypropylene secondary and/or tertiary cross-linkers having vinyl ketone, diallyl ether or divinyl ether terminal end groups, or of divinyl benzene, with acrylic, or methacrylic, such as maleic or itaconic monomers.